Abstract

Participation of E-cadherin in the Wnt signaling pathway was suggested
because of the dual role of β-catenin in cell adhesion and the Wnt
signaling cascade. Whereas β-catenin interacts at the cell membrane
with the cell adhesion protein E-cadherin, in the nucleus it activates
Wnt target genes through formation of transcriptionally active
complexes with members of the Tcf/Lef family of transcription factors.
Here, we analyzed by PCR and direct cycle sequencing 26 human breast
cancer cell lines for alterations in the E-cadherin
gene. Genetic alterations were identified in eight cell
lines. Five cell lines had truncating mutations, whereas three cell
lines had in-frame deletions in the gene transcript and expressed
mutant E-cadherin proteins at the cell membrane. Involvement of
E-cadherin in the Wnt pathway was evaluated through determination of
the activity of a Tcf reporter gene, which had been
transiently transfected into 15 breast cancer cell lines. None of six
E-cadherin mutant cell lines and four cell lines that
exhibit transcriptional silencing of the E-cadherin gene
showed Tcf-mediated transcriptional activation.
E-cadherin wild-type cell line DU4475 exhibited
constitutive Tcf-β-catenin signaling activity and was found to
express truncated APC proteins. These results indicate that if cellular
transformation occurred through mutation of E-cadherin,
it is not mediated via constitutive activation of the Wnt signaling
pathway.

The APC tumor suppressor gene was found to be mutated in a
majority of colorectal carcinomas (reviewed in Refs.
25
and
26
). The genetic alterations generally resulted in
truncated APC proteins that were no longer able to interact withβ
-catenin and axin. Consequently, inactivation of APC leads to
nuclear translocation of β-catenin, where it complexes with Tcf4 and
inappropriately activates the transcription of target genes
(27)
. In colorectal cancers with wild-type APC
genes
(28)
, in melanoma
(29)
, and in some
other cancer types (reviewed in Refs.
30
and
31
), dominant mutations in the
β-catenin (CTNNB1) gene have been
identified. These mutations affect the GSK-3β phosphorylation sites
of β-catenin and were shown to lead to stable Tcf-β-catenin
complexes in the nucleus and to constitutive transcriptional activation
of target genes
(28)
. Mutations in the AXIN1
and AXIN2 genes have recently been identified in
hepatocellular carcinomas
(32)
and in mismatch
repair-defective colorectal cancers
(33)
, respectively.
These mutations all predicted truncated axin proteins, which would be
expected to abolish proper degradation of β-catenin and result in
inappropriate activation of Tcf target genes.

E-cadherin is a transmembrane glycoprotein that mediates
calcium-dependent cell adhesion between epithelial cells (reviewed in
34
and
35
). Through its extracellular
calcium-binding domains, E-cadherin forms a molecular zipper with other
E-cadherin proteins that are located in the adherens junctions between
adjacent cells. The intracellular domain of E-cadherin interacts with
the actin cytoskeleton via a protein complex containing α-catenin,β
-catenin, and γ-catenin
(36,
37,
38)
. Tyrosine
phosphorylation of β-catenin in v-Src-
(39)
or Ras-
(40)
transformed cells or in cells
stimulated with epidermal growth factor
(41)
induced
release of the E-cadherin-catenin complex from the cytoskeleton and
diminished cell adhesion. As a result, the cells had lost their
characteristic epithelial growth pattern and had adopted a
fibroblast-like cell morphology.

Loss of E-cadherin expression or function has been implicated in tumor
invasion and metastasis (reviewed in Ref.
42
).
E-cadherin-mediated cell adhesion seemed to be crucial in the
transition from adenoma to carcinoma
(43)
, and
reconstitution of human
(44,
45,
46)
, canine
(47)
, murine
(48)
, or rat
(49)
cancer cell lines with wild-type E-cadherin cDNA was shown
to reverse invasiveness of the cancer cells. Primary tumor specimens of
various human cancer types were frequently found to exhibit aberrant
E-cadherin protein expression and, consistent with the experimental
models, this was often associated with the invasiveness of the tumors
(reviewed in Ref.
50
). Alterations in the
E-cadherin (CDH1) tumor suppressor gene have been
identified in nearly one-half of lobular breast carcinomas and
diffuse-type gastric carcinomas and in a small proportion of
gynecological cancers (reviewed in Refs.
51
and
52
).

The role of E-cadherin as a tumor suppressor protein and the dual role
of its binding partner β-catenin in cell adhesion and Wnt signaling
could indicate a function for E-cadherin in the Wnt pathway. In the
absence of an appropriate Wnt signal, for example, E-cadherin might
sequester β-catenin at the cell membrane, thereby preventing the
formation of Tcf-β-catenin complexes in the nucleus. Mutation of
E-cadherin in cancer cells may disrupt the interaction withβ
-catenin, thereby promoting nuclear translocation of β-catenin and
inappropriate formation of transcriptionally active Tcf-β-catenin
complexes. E-cadherin-null embryonic stem cells had indeed been
reported to exhibit Lef-β-catenin-mediated transcriptional
activation, which was antagonized by transient expression of wild-type
E-cadherin cDNA
(53)
.

If E-cadherin is indeed a participant of the Wnt pathway, genetically
mutant E-cadherin cancer cells should have constitutive Tcf-mediated
transactivation, similar to mutant APC, β-catenin, or axin cancer
cells
(27, 28, 32, 33)
. To test this hypothesis, we
performed a mutational analysis of E-cadherin in 26 human breast cancer
cell lines and identified 8 cell lines with genetic alterations in
E-cadherin. The involvement of E-cadherin in the Wnt
signaling pathway was evaluated through determination of
transcriptional activation of a Tcf-responsive Luciferase
reporter gene in 15 cell lines, including 6 E-cadherin
mutant cell lines and 4 cell lines with transcriptional silencing of
the E-cadherin gene.

PCR.

Genomic DNA was extracted using the Qiagen DNeasy kit. Exonic sequences
of E-cadherin (GenBank accession no. Z13009) were amplified
using primers designed to anneal to bordering intron sequences
(54)
. Amplification of genomic DNA (20 ng) was performed
by 2 min at 94°C and then by 35 cycles of 30 s at 94°C, 1 min
at the appropriate annealing temperature, and 1 min at 72°C, with a
final extension of 5 min at 72°C. PCR was performed in 10
mm Tris (pH 9.0) 50 mm KCl,
0.1% Triton X-100, 1.5 mm
MgCl2, 200 μm of each
deoxynucleotide triphosphate, and 0.75 units Taq Polymerase (Promega)
in a final volume of 15 μl. Exons 4 and 5, as well as exons 8 and 9,
were concurrently amplified. For exons 12–16, 0.5
m Betaine and 1% DMSO were included in the
reactions.

Microsatellite markers D16S421, D16S496,
D16S2621, and D16S2624 were used to PCR-amplify
polymorpic loci from the genome of the breast cancer cell lines. PCR
was performed essentially as described above, except that the reactions
were radioactively labeled with [α-32P]-dATP,
and 5 ng template DNA was used.

Reverse transcription-PCR.

RNA was extracted using the Qiagen RNeasy kit. First-strand synthesis
was performed using Ready-to-go-you-prime-first-strand-beads
(Pharmacia). E-cadherin transcripts were then amplified by
PCR, using E-cadherin-specific primers that annealed to
sequences ∼100 bases upstream of the first and downstream of the last
codon.

Cycle Sequencing.

E-cadherin amplification products were incubated with 10
units Exonuclease I and 2 units shrimp alkaline phosphatase
(United States Biochemical Corp.) for 15 min at 37°C, and the enzymes
were then inactivated for 15 min at 80°C. One-tenth of the reaction
was sequenced by 60 cycles of 30 s at 94°C, 30 s at the
appropriate annealing temperature, and 1 min at 72°C using conditions
recommended by the manufacturer (Thermo Sequenase Cycle Sequencing kit;
Pharmacia).

Western Blotting.

Cell lysates were prepared by resuspension of cells in Laemni sample
buffer and subsequent boiling for 5 min. Proteins were separated by
electrophoresis in a 3% low-melting point agarose gel and transferred
to Hybond-P membranes (Amersham) by overnight capillary blotting. Blots
were incubated with anti-APC antibody FE9 (Oncogene Research Products)
and then by horseradish peroxidase-conjugated rabbit antimouse
secondary antibodies (DAKO). Reactions were visualized through enhanced
chemiluminescence (ECL; Amersham).

Reporter Gene Assays.

Tcf reporter constructs pTOPGLOW and pFOPGLOW were generated by
insertion of SalI-fragments from the pTOP/FOPFLASH reporter
constructs that contain a multimerized Tcf-binding motif
(27)
into the SalI-linearized p19TATA-luc
vector that contains a minimal E1b TATA box upstream of the
Firefly Luciferase cDNA.

For transient transfections, cells were grown in RPMI 1640 containing
10% FCS, to 50–80% confluency in six-well plates. Cells were
transfected with 1 μg of pTOPGLOW or pFOPGLOW using Fugene-6
(Boehringer Mannheim) in the presence of 10% serum. Transfection
efficiencies were determined by cotransfection of 100 ng of pRL-TK
reporter construct (Promega) that contained the Renilla
Luciferase cDNA under control of the herpes simplex virus
thymidine kinase promotor. Cells were harvested 24 h after
transfection, washed with PBS, and resuspended in Passive Lysis buffer
(Promega). Activities of Firefly and Renilla luciferases were measured
sequentially from a single sample using the Dual-Luciferase Reporter
Assay System (Promega) on a Lumat LB9507 luminometer. Tcf-mediated gene
transcription was defined by the ratio of pTOPGLOW:pFOPGLOW luciferase
activities, where the luciferase activity of the internal control
reporter pRL-TK was used to correct for differences in transfection
efficiency.

RESULTS

E-cadherin Mutational Analysis.

Twenty-six human breast cancer cell lines (Table 1)
⇓
were analyzed for
allelic loss of chromosome 16q22 by PCR using microsatellite markers
D16S421, D16S496, D16S2621, and
D16S2624, which were located in the chromosomal region
encompassing the E-cadherin gene. Analysis of genomic DNA
from 25 unrelated, randomly selected individuals revealed
heterozygosity ratios of 0.36 for marker D16S421, 0.40 for
D16S496, 0.88 for D16S2621, and 0.76 for
D16S2624. Allelic loss at 16q22 was presumed when a cell
line had a single allele size at each of the four loci with a
P = 0.01 (i.e., the probability
that a heterozygous sample had a single allele size at each locus).
Fifteen of 26 (58%) breast cancer cell lines were considered to have
loss of heterozygosity at 16q22 (Table 1)
⇓
. None of the 25 DNAs from the
unrelated control individuals were homozygous at all four loci,
validating the statistical approach.

The coding sequence of E-cadherin was analyzed for genetic
alterations in 24 breast cancer cell lines by PCR and by direct cycle
sequencing. Homozygous deletions of E-cadherin gene
sequences were identified in four breast cancer cell lines (Table 1)
⇓
.
Cell line MDA-MB-134VI had a deletion of exon 6 of the
E-cadherin gene, cell line MPE600 had a deletion of exon 9,
and cell line OCUB-F had a deletion of exon 2. Cell line SK-BR-3 had a
homozygous deletion of exons 2 through 12, but had retained exon 1 and
exons 13–16 (Fig. 1A)
⇓
. All homozygous deletions were confirmed by duplex PCR
using an unrelated primer pair [DPC2′, 500-bp fragment;
(55)]
. Exons 2–16 of E-cadherin were
successfully amplified from the genome of the remaining 20 breast
cancer cell lines. Amplification products of exon 1 generally contained
fragments of various lengths, likely attributable to primer annealing
at homologous sequences in the human genome. Exon 1 was excluded from
additional analysis.

The amplified E-cadherin sequences were analyzed for
alterations by cycle sequencing, using the intronic PCR primers or
outwardly directed exonic primers. Alterations in the
E-cadherin gene sequence were identified in four breast
cancer cell lines (Table 1)
⇓
. Cell line CAMA-1 had an agGTT→aaGTT
alteration in the splice acceptor site of exon 12 (Fig. 1B)
⇓
,
cell line EVSA-T had an ACTgtaa→ACTaa alteration in the splice donor
site of exon 5, and cell line SK-BR-5 had an agATC→acATC alteration
in the splice acceptor site of exon 5. Cell line SUM44PE had deleted a
thymidine residue at codon 423, exon 9 (ATTTGT→ATTGT). All sequence
alterations were confirmed by sequencing of an independently amplified
template. The mutational analysis data are summarized in Table 1
⇓
.

E-cadherin Expression Analysis.

E-cadherin protein expression was analyzed by immunohistochemistry
using antibody TL#36 directed against a COOH-terminal epitope (residues
735–883) of E-cadherin. E-cadherin was found to be expressed mainly at
the cell membrane in cell lines BT20, DU4475, MCF-7, MDA-MB-361,
MDA-MB-468, and T47D, that had a wild-type E-cadherin gene
sequence (Fig. 2
⇓
and Table 1
⇓
).

E-cadherin mutant cell lines CAMA-1, EVSA-T, and MPE600,
expressed the protein at the cell membrane and weak diffuse in the
cytoplasm (Fig. 2
⇓
and Table 1
⇓
). Protein expression was in concordance
with sequence analysis of E-cadherin transcripts, which
revealed in-frame deletions in all three cell lines. CAMA-1 had deleted
exon 11 and the first base of exon 12, where the mutated base in the
splice site apparently formed a new acceptor site with the first base
of exon 12. EVSA-T had deleted the last 42 bases of exon 5, where the
first two bases of this deletion apparently served as a new donor
splice site. Cell line MPE600 had skipped exon 9.

The COOH-terminal antibody TL#36 did not detect E-cadherin protein
expression in the E-cadherin mutant cell lines MDA-MB-134VI,
OCUB-F, SK-BR-3, SK-BR-5, and SUM44PE (Fig. 2
⇓
and Table 1
⇓
). Truncations
of the E-cadherin protein in these mutant cell lines were confirmed by
shifts in the reading frame of E-cadherin transcripts from
OCUB-F and SUM44PE. OCUB-F had deleted exon 2, resulting in a stop
codon immediately after the deletion, and the 423delT alteration in
SUM44PE predicted the addition of seven new amino acids and then a stop
codon.

Cell lines BT549, Hs578T, MDA-MB-231, and MDA-MB-435S did not have
detectable E-cadherin protein expression (Fig. 2
⇓
and Table 1
⇓
). The
latter three cell lines had been reported to have silenced
E-cadherin gene expression through methylation of CpG
islands in the promotor region
(69)
.
Methylation-associated silencing in BT549 was inferred from its
fibroblast-like growth pattern, which was typical for all cell lines
with silenced E-cadherin
genes.
5

β-Catenin protein expression patterns were similar to those of
E-cadherin in most cell lines; that is, the three E-cadherin mutants
with in-frame deletions and the E-cadherin wild-type cell lines
expressed both proteins in a comparable pattern, whereas the five
mutants with truncated E-cadherin proteins did not have detectable
expression of either protein (Fig. 2
⇓
and Table 1
⇓
). The four cell lines
that had methylation-associated silencing of E-cadherin
seemed to have normal β-catenin expression as judged by
immunofluorescence microscopy. Furthermore, cell line DU4475 hadβ
-catenin protein expression at the cell membrane in all cells and
nuclear β-catenin expression in about one-half of the cells.
Reactions with isotype-matched control antibodies were negative for all
cell lines. Representative examples of the immunohistochemical analysis
are shown in Fig. 2
⇓
.

Tcf-β-catenin Reporter Gene Assays.

Transcriptional activation mediated by Tcf-β-catenin protein
complexes was determined in 15 breast cancer cell lines, including 6
cell lines that had genetic alterations of E-cadherin and 4
cell lines that had methylation-associated silencing of the gene (Fig. 3
⇓
and Table 1
⇓
). Cells were transiently transfected with either the
pTOPGLOW or pFOPGLOW reporter constructs, which contained multimerized
wild-type or mutant Tcf-binding motifs upstream of the Firefly
Luciferase cDNA driven by a minimal E1b TATA box together
with the pRL-TK internal control reporter construct that contains the
Renilla Luciferase cDNA driven by the HSV-TK
promotor. Tcf-mediated gene transcription was defined by the
ratio of pTOPGLOW:pFOPGLOW luciferase activity after 24 h, each
corrected for luciferase activities of the pRL-TK reporter, where no
transcriptional activation equals 1.

Tcf-mediated transcriptional activation in breast cancer.
A, constitutive transcriptional activation was detected
in cell line DU4475 with the Tcf reporter construct pTOPGLOW but not
with the mutant construct pFOPGLOW. APC mutant colorectal cancer cell
line SW480 served as a positive control. Tcf-mediated transcriptional
activity was defined as the ratio of pTOPGLOW:pFOPGLOW luciferase
activities, each corrected for pRL-TK luciferase activities and where
no transactivation equals 1. B, expression of truncated
APC proteins in cell line DU4475, identified by Western blot analysis
using FE9 antibody. Left Lane, APC wild-type cells;
right Lane, cell line DU4475.

Cell line DU4475, which had a wild-type E-cadherin gene
sequence, showed a 300-fold increase in transcriptional activity of the
pTOPGLOW reporter as compared with the negative control pFOPGLOW. None
of the other 14 breast cancer cell lines had enhanced transcription of
the TOPGLOW reporter (Table 1
⇓
and Fig. 3A⇓
). As a control,
the APC mutant colorectal cancer cell line SW480 had a
600-fold enhanced transcriptional activity of pTOPGLOW as compared with
pFOPGLOW
(27)
. All reporter gene assays were performed at
least twice in duplicate transfections.

Possible aberrations of APC proteins in cell line DU4475 were addressed
by Western blot analysis. Expression of truncated APC proteins
(Mr150,000–200,000) but not
wild-type APC was detected with monoclonal antibody FE9, which is
directed against an NH2-terminal epitope of APC
(Fig. 3B)
⇓
.

DISCUSSION

E-cadherin Alterations in Breast Cancer.

Mutational analysis of the E-cadherin gene in 26 human
breast cancer cell lines revealed genetic alterations in 8 cell lines.
Although four of these mutant cell lines had been described previously
(70)
, our characterization revealed some novel aspects.
Whereas the E-cadherin gene sequence of cell line CAMA-1 was
reported to be wild-type
(70)
, we identified a
point-mutation in the splice acceptor site of exon 12 (Fig. 1B)
⇓
. Our analysis of E-cadherin transcripts in
CAMA-1, as well as in MPE600, revealed in-frame deletions in both cell
lines. Immunohistochemistry using an antibody directed against a
COOH-terminal epitope of E-cadherin revealed abundant expression of the
mutant proteins (Fig. 2)
⇓
, excluding the amplification of aberrant
transcripts. The E9 antibody that was used by Hiraguri et
al.(70)
may have failed to detect protein expression
in CAMA-1 and MPE600 because it is directed against an extracellular
epitope of E-cadherin that might have been affected in the mutant cell
lines.

Three of 8 E-cadherin mutant breast cancer cell lines had in-frame
deletions of gene sequences, whereas mutational analyses of primary
breast carcinomas
(54, 71)
had identified in-frame
deletions in only 2 of 27 mutant tumors. This discrepancy is not
readily explained by differences in experimental procedures (PCR
versus SSCP) but possibly reflects biases in either the cell
line collection or in the sampling of primary tumor specimens. In this
respect, it should be noted that in-frame deletions were the
predominant E-cadherin gene alterations identified in
primary gastric carcinomas
(51, 52)
.

Alterations in the E-cadherin gene had been identified in
nearly one-half of lobular breast carcinomas
(54, 71)
, a
histological subtype that represents 10–20% of primary breast
carcinomas. The relatively high percentage of E-cadherin
mutants in our cell line collection (8 of 26; 31%) may be attributable
to the homozygous deletion of gene sequences in one-half of the mutant
cell lines. Homozygous deletions would not, however, be detected
in primary tumor specimens because of the inevitable presence of
non-neoplastic cells in these samples. Four breast cancer cell lines
had no detectable E-cadherin protein expression because of
methylation-associated transcriptional silencing. Together with the 8
genetic E-cadherin mutants, 12 of 26 (46%) breast cancer
cell lines had aberrant E-cadherin protein expression, a frequency that
is in concordance with ample immunohistochemical analyses of primary
breast carcinomas
(50)
.

Interestingly, of four E-cadherin mutant breast cancer cell lines with
known histological subtype, only one was of lobular origin and
three were of ductal origin (Table 1)
⇓
. Also, all four cell lines with
silenced E-cadherin genes were of ductal origin. This is in
contrast to genetic analyses on primary breast carcinomas, where mutant
E-cadherin genes were identified exclusively in cancers of
lobular histology
(54, 71)
. The reason for this
discrepancy is presently unclear, but it may again reflect biases in
these tumor collections.

The Wnt Pathway in Breast Cancer.

Int-1/Wnt-1 was originally identified as an oncogene that
had been activated in murine breast carcinomas through mouse mammary
tumor virus integrations
(72, 73)
. Furthermore, Tcf4, the
Tcf/Lef family member that is involved in the inappropriate Wnt pathway
activation in colorectal cancer, is specifically expressed in epithelia
of the intestine and mammary gland
(74)
. It was,
therefore, perhaps somewhat surprising that we detected Tcf-mediated
transcriptional activation in only 1 of 15 breast cancer cell lines.
This cell line, DU4475, was found to express truncated APC proteins and
had nuclear β-catenin protein expression, both consistent with Wnt
pathway activation. These results were confirmed by a recently reported
mutational analysis of Wnt pathway members in 24 breast cancer cell
lines, where DU4475 was also identified as the only cell line with a
mutation in the APC gene (E1577stop; Ref.
75
).
The low mutation frequency of APC in breast cancer cell lines is
similar to that reported for primary breast carcinoma specimens (2 of
31; 6%; Ref.
76
). Our results thus confirm that
inactivation of APC is rare in breast cancer, but they also indicate
that inappropriate activation of the Wnt pathway through mutation of
other members of the signaling cascade is an uncommon event in breast
carcinogenesis. Because activation of the Wnt pathway is a prerequisite
for evasion of tumor suppressive mechanisms in the intestine, the
biology of breast cancers appears, in this respect, to be quite
distinct from that of colorectal cancers.

E-cadherin and the Wnt pathway.

None of six genetically mutant E-cadherin breast cancer cell
lines, and four cell lines with transcriptionally silenced
E-cadherin genes exhibited Tcf-mediated transcriptional
activation. These results indicate that mutant E-cadherin tumor
suppressor proteins do not constitutively activate the Wnt pathway, and
thus do not resemble mutant APC, β-catenin, or axin proteins in
colorectal and hepatocellular cancer
(27, 28, 32, 33)
.
Similar results were recently reported for human breast cancer cell
lines that had lost E-cadherin protein expression
(77)
.
Direct evidence that E-cadherin is not involved in the Wnt pathway,
however, had not been provided because the absence of E-cadherin
expression had not been substantiated by mutations in the
E-cadherin gene. Notably, E-cadherin expression in these
cell lines might have been silenced in association with methylation of
CpG islands in the E-cadherin promotor region. This
epigenetic mechanism of inactivation is not yet fully understood, but
it has been shown to be heterogeneous and changing dynamically in human
breast cancers
(78)
and therefore disputable would lead to
constitutive activation of Wnt signaling in the cancer cells. The
observed Tcf-β-catenin-signaling activity in E-cadherin-null
embryonic stem cells
(53)
seems in contrast with these and
our results and may reflect an inherently reduced efficiency of
embryonic stem cells to degrade free β-catenin proteins. Sanson
et al.(79)
elegantly demonstrated that the
function of E-cadherin in cell adhesion does not affect Wnt signaling
in Drosophila. It is, however, entirely possible that the
E-cadherin function that is abrogated through E-cadherin
gene mutations in human cancers is distinct from its function in cell
adhesion, and E-cadherin could thus still be involved in the Wnt
pathway.

Here, we conclusively excluded E-cadherin as a participant of the Wnt
pathway through extensive analysis of natural E-cadherin
mutants with in-frame deletions, truncating mutations, or
methylation-associated silenced E-cadherin genes. The five
mutant breast cancer cell lines with truncated E-cadherin proteins
would be expected to be incapable of sequestering β-catenin at the
cell membrane, because these mutant proteins have deleted the
COOH-terminal β-catenin interaction site. Indeed, none of these five
truncation mutants expressed β-catenin at the cell membrane, but,
surprisingly, neither did they have detectable β-catenin expression
elsewhere in the cell (Fig. 2)
⇓
. Normal activity of the APC destruction
complex is likely to prevent any significant increase in levels of freeβ
-catenin in these E-cadherin truncation mutants. Release ofβ
-catenin from the cell membrane is thus in itself not sufficient to
lead to transcriptionally active Tcf-β-catenin complexes in the cell
nucleus. The apparently normal expression of β-catenin proteins in
the three breast cancer cell lines with in-frame deletions of
E-cadherin reinforces that abolishment of β-catenin sequestering does
not bear functional significance in the E-cadherin tumor suppressive
pathway.

Acknowledgments

We thank Cor Breukel and Riccardo Fodde for their expert
assistance with APC Western blotting. We also thank Silvia van der
Flier, Ruud van Gurp, Pieter Jaap Krijtenburg, and Frank van de Panne
for technical advice.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

↵1 This work was supported by the Dutch Cancer
Society Koningin Wilhelmina Fonds, Grant DDHK 97-1644.
Additional support was received from the De Kock Society and the
Nijbakker-Morra Society.